Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • @mastal

    Thank you for the quick reply! Unfortunately, it's still displaying the same error message. But at least I know the problem is around there, so that's good news.

    So, could there be a way to possibly direct trimmomatic to the file without beginning with C:? Or could this possibly be another issue?

    I've tried other symbols like brackets and parenthesis just in case 'C:' wasn't it. Same message.

    Comment


    • Hi

      sorry for the inconvenience. At the moment it might be best to not have to use C: at all as trimmomatic looks for data after :
      E.g. having the clip file in the actual directory - yukk.

      Best Wishes
      Björn

      Comment


      • I figured it would come to that. I think my best option would be to use Linux and not have to direct trimmomatic using "C:". Don't file paths in Linux all begin with a "/home/..."? I'll try that and see if it works.

        Comment


        • Originally posted by kevluv93 View Post
          I figured it would come to that. I think my best option would be to use Linux and not have to direct trimmomatic using "C:". Don't file paths in Linux all begin with a "/home/..."? I'll try that and see if it works.
          No, not really. The path depends on the system... Java works fine in Windows, and as long as the classpath is set correctly, it should work fine anywhere. You just need to set the "-cp" variable correctly.

          Comment


          • Originally posted by mastal View Post
            What was the trimmomatic command that you used, and what sequences did you use in the adapters fasta file?
            java -jar trimmomatic-0.32.jar PE -threads 11 -trimlog trim_keepbothread.log Hiseq_1.fastq Hiseq_2.fastq Hiseq_1_keeppaired.fastq Hiseq_1_keepunpaired.fastq Hiseq_2_keeppaired.fastq Hiseq_2_keepunpaired.fastq ILLUMINACLIP:NexteraPE-PE.fa:2:30:10:8:TRUE LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

            And my adapter fasta file contain the transposase sequence and primer sequence provided by illumina:

            >Trans1_rc
            CTGTCTCTTATACACATCTGACGCTGCCGACGA
            >Trans2_rc
            CTGTCTCTTATACACATCTCCGAGCCCACGAGAC

            >Primerprefixi5_rc
            GACGCTGCCGACGA
            >Primerprefixi7_rc
            CCGAGCCCACGAGAC


            Thanks

            Comment


            • Could you guys give me an example script of setting up a classpath for "ILLUMINACLIP:" to get access to the adapter files in the trimmomatic binary download? I should probably mention that my experience with scripting languages begins and ends with "making stick figures in JavaScript", so forgive me if I'm asking for baby steps. I'm still using Windows 8 for this trimmomatic job (it's the OS that all the computers in our college use, so I don't really have a choice.)

              @ Bjorn, you mentioned "having the clip file in the actual directory", and that trimmomatic looks for documents after ":". With this info, I deleted the "C:" that came after ILLUMINACLIP: but I got an error message telling me, "ArrayIndexOutOfBoundsException: 1".

              @Brian Bushnell, you mentioned that trimmomatic will work on windows as long as I specify the classpaths to the files correctly.

              So, could someone give me an example of what I need to write after "ILLUMINACLIP:" to let "ILLUMINACLIP:" get the adapter sequence fa. files that are saved on my desktop? I'm just right clicking these icons and copy/pasting their locations into the trimmomatic script. How else does one make a path to a file?

              Thanks for the help! Any input is appreciated!

              Comment


              • kevluv,

                You are not doing anything wrong with Java; your command would fail in any environment. The classpath appears to be correct, you just have the wrong command line.

                Comment


                • Hi guys, seems that Trimmomatic is very useful and performs better that others.

                  The only thing I can not find, if present at all. Where can I get the info if any adaptor clipping appeared? Yes I can analyse the result by fastqc or go deep to log (still no info for clipping), but is there any obvious place to find adaptor clipping results?

                  The second question is - what is the clipping/trimming strategy applied for internal (junction Nextera adaptors)?

                  Comment


                  • could you please give a real example of using -baseout flag?
                    when using like this "... -phred33 PE S1R1.fatsq S1R2.fastq -baseout outS1.fasta ILLUMINACLIP..." it is still trying to find the list on names afterwards...

                    Comment


                    • Haha! I finally got it to work! It was just some user error on my part.

                      Changes:
                      Don't use "C:" at the beginning of files.
                      It works fine with Windows 8
                      I erased the ".fq" at the end of my input files, I thought I was supposed to specify what file type my input data was, but don't. The name of the file's good enough.

                      Worked like a charm, it was just user error on my part. Thanks for the help!

                      Comment


                      • great that it works now.

                        Comment


                        • Hi i have used trimmomtaic to clip the adapter sequences. Yet i am still having a problem with kmers, sequence per base content. My question is how do we improve the quality if there is any failure in Fastqc results????

                          Comment


                          • Hi, back again. When I use trimmomatic I get abnormally high Kmer reads on FastQC, I read out the Kmers and realized that most of my forward adapter was still inside of my cDNA.

                            I opened the TruSeq2 adapter file and realized that the Prefix PE/1 adapter (I guess that means the forward adapter?) Didn't match the forward adapter I was using, which is:
                            TruSeq Adapter, Index 2
                            5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGATGTATCTCGTATGCCGTCTTCTGCTTG

                            So I went in and replaced the default forward adapter with the sequence you see above.

                            Now I have another issue, for some reason trimmomatic has managed to cut 8GB of data into 2GB of data. (if I combine the forward and reverse paired and unpaired files) Fastqc is giving me Kmers that are similar to my sequence, and when I opened the forward paired file I saw that fairly large chunks of my primers are still at the 3' end of my cDNA.

                            ex.
                            GATCGGAAGAGCACACGTCTGAACTCCAGTCAC
                            GATCGGAAGAGCACACGTCTGAACTCC
                            ATCGGAAGAGCACACGTCTGAACTCCAGTCACCGA

                            They're just small enough for trimmomatic to miss. Is there any helpful suggestions you can give me regarding the settings of trimmomatic to help me cut these small bits of adapters off the end of my reads? Is there a reason why only small fragments of my adapter sequences would be left after using trimmomatic? Finally, is it usual for such a large portion of data to get cut when using trimmomatic or am I screwing this up? (8GB to 2GB of data)

                            Comment


                            • Originally posted by kevluv93 View Post
                              Hi, back again. When I use trimmomatic I get abnormally high Kmer reads on FastQC, I read out the Kmers and realized that most of my forward adapter was still inside of my cDNA.

                              I opened the TruSeq2 adapter file and realized that the Prefix PE/1 adapter (I guess that means the forward adapter?) Didn't match the forward adapter I was using, which is:
                              TruSeq Adapter, Index 2
                              5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGATGTATCTCGTATGCCGTCTTCTGCTTG
                              This sequence looks like a TruSeq-3 adapter - if so, either the TruSeq3-PE or TruSeq3-PE-2 adapter files should remove the sequencing adapters. The useful part of the reads should survive if they the adapters are in the normal position.

                              If you do have any surviving adapters, can you post a few examples?

                              Thanks,

                              Tony.

                              Comment


                              • Hey guys,
                                i`ve got some problems with Trimmomatic:

                                I have the following 100bp long read:

                                @HWI-ST365:34625ECACXX:5:1101:3183:2046 1:N:0:TGACCA
                                NCAGGGGGAACAGGCTGATCTCCCCCAAGAGTCCACATCGACGGGGAGGTTTGGCACCTCGATGTCGGCTCATCGCAACCTGGGGCGGAAGGACGTCCCC
                                +
                                #11A?DDDHHBBF?FHBGCGHGGDD@FFDDD9B@FHD8)<FHIG8B89>(,5,5(::<CB?'9@BC8;5?##############################

                                I only run SLIDINGWINDOW:4:15 on this read

                                and what i get is:

                                Log-File:

                                HWI-ST365:34625ECACXX:5:1101:3183:2046 1:N:0:TGACCA 52 0 52 48

                                trimmed fastq:

                                @HWI-ST365:34625ECACXX:5:1101:3183:2046 1:N:0:TGACCA
                                NCAGGGGGAACAGGCTGATCTCCCCCAAGAGTCCACATCGACGGGGAGGTTT
                                +
                                #11A?DDDHHBBF?FHBGCGHGGDD@FFDDD9B@FHD8)<FHIG8B89>(,5


                                But when i run my own script on the read i can see that the pattern (,5, beginning at position 50 has an average quality of 12.25, which is below the required 15. So the read should survive from position 1 to 49 and not until position 52 as determined by Trimmomatic.

                                So can anyone tell me where i am wrong?

                                Thanks
                                Mchicken

                                Comment

                                Latest Articles

                                Collapse

                                • seqadmin
                                  Strategies for Sequencing Challenging Samples
                                  by seqadmin


                                  Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
                                  03-22-2024, 06:39 AM
                                • seqadmin
                                  Techniques and Challenges in Conservation Genomics
                                  by seqadmin



                                  The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

                                  Avian Conservation
                                  Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
                                  03-08-2024, 10:41 AM

                                ad_right_rmr

                                Collapse

                                News

                                Collapse

                                Topics Statistics Last Post
                                Started by seqadmin, Yesterday, 06:37 PM
                                0 responses
                                11 views
                                0 likes
                                Last Post seqadmin  
                                Started by seqadmin, Yesterday, 06:07 PM
                                0 responses
                                10 views
                                0 likes
                                Last Post seqadmin  
                                Started by seqadmin, 03-22-2024, 10:03 AM
                                0 responses
                                51 views
                                0 likes
                                Last Post seqadmin  
                                Started by seqadmin, 03-21-2024, 07:32 AM
                                0 responses
                                68 views
                                0 likes
                                Last Post seqadmin  
                                Working...
                                X